Bottom Line:
We show that this single, evolutionary conserved exon defines an autonomous structural unit that, despite the minimal structural context, closely matches the structure of the same region in the entire receptor binding module.In eukaryotic genomes, exon and domain boundaries usually coincide.We report a case study where this assertion does not hold, and show that the autonomously folding, structural unit is delimited by exon boundaries, rather than by predicted domain boundaries.

Background: Notch signaling drives developmental processes in all metazoans. The receptor binding region of the human Notch ligand Jagged-1 is made of a DSL (Delta/Serrate/Lag-2) domain and two atypical epidermal growth factor (EGF) repeats encoded by two exons, exon 5 and 6, which are out of phase with respect to the EGF domain boundaries.

Results: We determined the 1H-NMR solution structure of the polypeptide encoded by exon 6 of JAG1 and spanning the C-terminal region of EGF1 and the entire EGF2. We show that this single, evolutionary conserved exon defines an autonomous structural unit that, despite the minimal structural context, closely matches the structure of the same region in the entire receptor binding module.

Conclusion: In eukaryotic genomes, exon and domain boundaries usually coincide. We report a case study where this assertion does not hold, and show that the autonomously folding, structural unit is delimited by exon boundaries, rather than by predicted domain boundaries.

Figure 2: Solution structure of J1ex6. Backbone representation of 20 NMR models. The thickness of the trace is proportional to the backbone RMSD towards the mean. Cysteine residues are labeled with residue number and disulfide bonds are in yellow.

Mentions:
The solution structure of J1ex6 was determined by 1H NMR spectroscopy (PDB: 2KB9) (Table 1, Additional files 1 and 2). Disulfide bonds were experimentally determined by targeted proteolysis and MS analysis in a three-step strategy that lead to the unambiguous assignment of the disulfide topology, and they were explicitly used in structure calculations as distance constraints. The overall fold of J1ex6 is mainly dictated by the four disulfide bonds and lacks well defined secondary structure elements, as well as a true hydrophobic core (Figure 2). The mean pairwise RMSD values for the backbone and all heavy atoms (in parenthesis) are 1.04 ± 0.24 Å (1.65 ± 0.30 Å) from the first to the last half-cystine (residues C253–C264), 1.16 ± 0.39 Å (2.11 ± 0.52 Å) for the N-terminal overhang (residues 265–293), and 0.71 ± 0.24 Å (1.10 ± 0.26 Å) for the core EGF2 repeat (residues C265–C293). The distribution of psi/phi angles in the Ramachandran map for the 20 selected models is: 50.2% in most favored regions, 47.0% in additionally allowed regions, 2.9% in generously allowed regions, and 0.0% in disallowed regions. Whereas the availability of heteronuclear NMR data would have probably improved the precision of the models, the results for the psi/phi distribution are in line with the statistics for a set of 49 NMR structures of single and tandem EGF repeats deposited at the PDB (data not shown). It is thus possible that the sub-optimal distribution of psi/phi angles in the Ramachandran map of EGF repeats is a consequence of the constraints dictated by the disulfide bonds.

Figure 2: Solution structure of J1ex6. Backbone representation of 20 NMR models. The thickness of the trace is proportional to the backbone RMSD towards the mean. Cysteine residues are labeled with residue number and disulfide bonds are in yellow.

Mentions:
The solution structure of J1ex6 was determined by 1H NMR spectroscopy (PDB: 2KB9) (Table 1, Additional files 1 and 2). Disulfide bonds were experimentally determined by targeted proteolysis and MS analysis in a three-step strategy that lead to the unambiguous assignment of the disulfide topology, and they were explicitly used in structure calculations as distance constraints. The overall fold of J1ex6 is mainly dictated by the four disulfide bonds and lacks well defined secondary structure elements, as well as a true hydrophobic core (Figure 2). The mean pairwise RMSD values for the backbone and all heavy atoms (in parenthesis) are 1.04 ± 0.24 Å (1.65 ± 0.30 Å) from the first to the last half-cystine (residues C253–C264), 1.16 ± 0.39 Å (2.11 ± 0.52 Å) for the N-terminal overhang (residues 265–293), and 0.71 ± 0.24 Å (1.10 ± 0.26 Å) for the core EGF2 repeat (residues C265–C293). The distribution of psi/phi angles in the Ramachandran map for the 20 selected models is: 50.2% in most favored regions, 47.0% in additionally allowed regions, 2.9% in generously allowed regions, and 0.0% in disallowed regions. Whereas the availability of heteronuclear NMR data would have probably improved the precision of the models, the results for the psi/phi distribution are in line with the statistics for a set of 49 NMR structures of single and tandem EGF repeats deposited at the PDB (data not shown). It is thus possible that the sub-optimal distribution of psi/phi angles in the Ramachandran map of EGF repeats is a consequence of the constraints dictated by the disulfide bonds.

Bottom Line:
We show that this single, evolutionary conserved exon defines an autonomous structural unit that, despite the minimal structural context, closely matches the structure of the same region in the entire receptor binding module.In eukaryotic genomes, exon and domain boundaries usually coincide.We report a case study where this assertion does not hold, and show that the autonomously folding, structural unit is delimited by exon boundaries, rather than by predicted domain boundaries.

Background: Notch signaling drives developmental processes in all metazoans. The receptor binding region of the human Notch ligand Jagged-1 is made of a DSL (Delta/Serrate/Lag-2) domain and two atypical epidermal growth factor (EGF) repeats encoded by two exons, exon 5 and 6, which are out of phase with respect to the EGF domain boundaries.

Results: We determined the 1H-NMR solution structure of the polypeptide encoded by exon 6 of JAG1 and spanning the C-terminal region of EGF1 and the entire EGF2. We show that this single, evolutionary conserved exon defines an autonomous structural unit that, despite the minimal structural context, closely matches the structure of the same region in the entire receptor binding module.

Conclusion: In eukaryotic genomes, exon and domain boundaries usually coincide. We report a case study where this assertion does not hold, and show that the autonomously folding, structural unit is delimited by exon boundaries, rather than by predicted domain boundaries.